Long-range High-resolution Mapping of Chromatin

染色质的远程高分辨率绘图

• 批准号: 6943147
• 负责人: ROBERT KINGSTON
• 金额: $ 43.25万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6943147
• 关键词: biotechnology; cell line; chromatin; genetic mapping; high throughput technology; molecular biology information system; stem cells; technology /technique development

DESCRIPTION (provided by applicant): Every known nuclear regulatory event that occurs on the genome appears to be associated with a change in chromatin structure. Regulatory sequences that specify transcription, replication, DNA repair and recombination are located in regions whose structure changes with regulated function of these elements. The goal of this application is to devise technology that will allow chromatin structure to be examined over very large (100 kb or greater) regions of the genome. The focus is on mapping cleavage sites for chemicals and enzymes whose activity is known to display sensitivity to changes in chromatin structure. Development of this technology will not only provide an important, largely unbiased, mechanism for searching for novel regulatory elements, but will also provide a tool to increase the understanding of long-range changes in chromatin structure. There is precedent for the utility of mapping of cleavage sites in understanding gene regulation; for example the mapping of DNase hypersensitive sites has played a significant role in understanding promoter, enhancer and LCR function. A systematic, automated mapping of cleavage sites over regions of the genome an order of magnitude larger than those examined previously will prove to be a useful tool in both identification of regulatory elements and formation of hypotheses concerning the regulation of higher order chromatin structures. The following Aims will be pursued to develop a technology for long-range high-throughput mapping of cleavage sites: Aim 1 will develop a single tube protocol for mapping cleavage sites in single copy mammalian DNA and will automate that protocol; Aim 2 will apply the technology developed in Aim 1 to mapping large (up to 120 kb) regions of the mouse genome both in an undifferentiated multipotent stem cell line and in a homogeneous differentiated population of cells derived from that stem cell line. Aim 3 will develop bioinformatics tools to interpret the data collected in Aim 2.

描述（由申请人提供）：基因组上发生的每一个已知的核调控事件似乎都与染色质结构的变化相关。 指定转录、复制、DNA修复和重组的调节序列位于其结构随这些元件的调节功能而变化的区域中。 本申请的目标是设计技术，该技术将允许在基因组的非常大（100 kb或更大）的区域上检查染色质结构。 重点是绘制化学品和酶的切割位点，已知其活性对染色质结构的变化敏感。 这项技术的发展不仅将提供一个重要的，很大程度上是公正的，寻找新的调控元件的机制，但也将提供一个工具，以增加对染色质结构的长期变化的理解。 切割位点的作图在理解基因调控中的应用已有先例;例如，DNA酶超敏位点的作图在理解启动子、增强子和LCR功能中发挥了重要作用。 一个系统的，自动化的映射切割位点的基因组区域的一个数量级大于以前检查的将被证明是一个有用的工具，在两个监管元件的识别和形成的假说有关的调节更高级别的染色质结构。 目标1将开发一种用于绘制单拷贝哺乳动物DNA中的切割位点的单管方案，并将使该方案自动化;目标2将把目标1中开发的技术应用于绘制大型在未分化的多能干细胞系和来源于该干细胞系的均质分化细胞群中的小鼠基因组的多个（至多120 kb）区域。目标3将开发生物信息学工具，以解释目标2中收集的数据。